The goal of this proposal is to identify potential probiotic strains and their inhibitory mechanisms in order to develop new therapies to prevent periodontitis. The significance of this application relates to global concerns about widespread and increasing antibiotic resistance and the growing support for more ecologically acceptable therapies including pro- and prebiotic approaches using antagonistic bacteria or their products. The most frequently employed probiotic bacteria for gastrointestinal health are lactic acid bacteria (LAB), but little is known about their use in the oral cavity or how they might suppress pathogens. Even less is known about the potential probiotic effects of other non-LAB bacteria and these constitute significant knowledge gaps in the probiotic field. In our preliminary data, we have identified potential LAB and non-LAB oral commensal bacteria that can suppress the key oral pathogen Porphromonas gingivalis. In this proposal, we will use novel technologies to identify how these bacteria interact and explore their potential as probiotics. In Aim 1 we will identify genes involved in interactions between P. gingivalis and selected probiotic bacteria. A key innovation here is that we will utilize a recently developed saturated P. gingivalis transposon mutant library in co-culture with the probiotic bacteria and identify critical genes in the interacion by a massively parallel sequencing approach (Tn-seq) to quantitatively assess survival of mutants under selective pressure of probiotic activity. To complement identification probiotic target genes by Tn-seq, we will also determine gene expression patterns by RNA-seq in wild-type P. gingivalis cultured with and without probiotics to identify functions most affected. Aim 2 addresses identification of growth inhibiting molecules produced by selected probiotics, first by setting up a test system of targeted deletions and in trans complemented strains of P. gingivalis genes identified by Tn-seq and RNA-seq as highly affected by probiotic bacteria to confirm probiotic inhibition. Metabolites in supernatants of candidate probiotic strains will be identifiedby mass spectrometry and tested on the mutant and complemented mutant strains. In Aim 3 we will test combinations of P. gingivalis and either probiotic bacteria or synthetic inhibitor derivatives for activity in a 3-D gingival infection model to quantify survival of both P. gingivals and probiotics and measure markers of inflammation in gingival cells. The information obtained upon completion of this work will provide new insights into how metabolic actions between bacteria can be exploited in order to maintain a healthy microbiota. Inhibitory bacteria or their products identified in our project may be candidate probiotic agents for the prevention of periodontitis. Genes/pathways that are targets of prebiotics from oral commensal bacteria and LAB may also become lead targets for development of drugs that control P. gingivalis virulence.